Pure culture of Agrobacterium sp. which degrades ferric chelates

ABSTRACT

A novel microorganism has been isolated and identified as Agrobacterium sp., a gram-negative aerobe. The Agrobacterium sp. microorganisms has been found to efficiently degrade ferric chelates of aminopolycarboxylic acids, such as ethylenediaminetetraacetic acid and related compounds, found in aqueous waste solutions. A biologically pure culture of Agrobacterium sp. and a process for using the culture are disclosed.

This is a division of application Ser. No. 603,381 filed Oct. 26, 1990,now U.S. Pat. No. 5,252,483, which is a continuation of 07/507,931,filed Apr. 11, 1990 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to biological degradation of ferricchelates of aminopolycarboxylic acids. More particularly, the inventionrelates to the use of the microorganism Agrobacterium sp. to degradethese compounds, especially from aqueous solutions, as well as tobiologically pure cultures of Agrobacterium sp.

2. State of the Art

It has heretofore been recognized that metal chelates ofaminopolycarboxylic acids, such as ferricethylenediaminetetraacetic acid(ferric-EDTA) and other similar chelates, were not readily biodegraded.See, for example, Gerike et al., Ecotoxicology and Env. Safety,3:159-173 (1975).

This inability to readily degrade such metal chelates leads to numerousenvironmental concerns. In particular, in secondary treatment with anactivated sludge, the biodegradation of these metal chelates is so slowthat these chelates have been reported to pass through secondarytreatment facilities and discharged into the environment without notabledegradation. See Thom et al., Proc. Roy. Soc. of London, 189:347-357(1975). This has led to concerns for the possible bioaccumulation of thechelate in the environment as well as the possible transport of heavymetals by release of high concentrations into polluted rivers orsediments. Additionally, EDTA has also been implicated in themobilization of radionuclides from nuclear waste disposal sites.

On the other hand, accumulation of EDTA in the environment over timeperiods on the order of years has been shown to be unlikely. Forexample, EDTA contamination in soil, at relatively low concentrations(2-1000 micrograms per gram of soil), have been demonstrated to bebiologically removed. See Tiedje, Appl. Microbiol., 30:327-329 (1975)and Tiedje, J. Environ Qual. 6:21-26 (1977). In these studies, thedisappearance of low concentrations of EDTA and its metal chelates wasshown to occur in 15-45 weeks on a wide variety of soils. However, nosingle bacterial type was isolated which was capable of metabolizingEDTA or its metal chelates.

At somewhat higher concentrations (<2 mM) in aqueous solution, Belly etal., Appl. Microbiol, 29:787-794 (1975) have investigated the aerobicbacterial degradation of the Fe⁺³ EDTA chelate by an acclimated mixedpopulation of bacteria. Disappearance of more than 90% of the substratein 5 days was observed at these concentrations. Again, no singlebacterial type was isolated which would grow on the ferric-EDTAsubstrate as sole carbon source.

The abstract for SU 525627 describes removal of heavy metal complexesfrom water by precipitation, using first a sodium hydroxide treatmentand then biological purification using aerobic microorganisms. Thefiltrate obtained from the sodium hydroxide treatment is purified withmicroorganisms at a pH of 8-9.

Japanese Laid-Open Patent Application (Kokai) No. 58-43782 states thatstrains of the genera Pseudomonas and Alcaligenes are capable ofdegrading EDTA under aerobic conditions. The maximum concentrationtested was 5 mM and required 5 days for 80% degradation.

Besides biological degradation, the ferric chelate of EDTA can bedegraded by ultraviolet irradiation. Specifically, under simulatedenvironmental conditions, Lockhart et al., Env. Sci. and Technol.,9:1035-1038 (1975) as well as Lockhart et al., Environ. Lett., 9:19-31(1975) have demonstrated degradation by the mechanism of UV irradiation.Rates of degradation in bright sunlight were found to vary from 1-2 daysfor removal of the parent compound at an initial concentration and pH of1.6 mM and 4.9, respectively, to greater than 10 days at an initialconcentration and pH of 9.8 mM and 6.9, respectively. In these studiestransformation of ferric-EDTA was demonstrated but extensivemineralization was not.

In view of the above, there is a continuing need in the art for anefficient method for the degradation of metal chelates ofaminopolycarboxylic acids, especially from industrial waste solutionscontaminated with such chelates. There is a further need in the art thatany such degradation process for the removal of such chelates beconducted rapidly without damaging the environment.

SUMMARY OF THE INVENTION

The present invention is directed to the discovery that themicroorganism Agrobacterium sp. (deposited with the American TypeCulture Collection, Rockville, Md., as ATCC No. 55002) rapidly andefficiently degrades ferric chelates of aminopolycarboxylic acids andaccordingly, this organism can be used to treat aqueous solutions so asto remove such ferric chelates.

Accordingly, in one of its composition aspects, the present invention isdirected to a biologically pure culture of the microorganismAgrobacterium sp., ATCC number 55002.

In another of its composition aspects, the present invention is directedto a biologically pure culture of Agrobacterium sp., which culture iscapable of degrading ferric chelates of aminopolycarboxylic acids froman aqueous solution containing one of more of the chelates.

In one of its method aspects, the present invention is directed to amethod of degrading ferric chelates of aminopolycarboxylic acids whichcomprises combining said chelates with a biologically pure culture ofAgrobacterium sp. This aspect of the invention further relates to amethod for degrading ferric chelates of ethylenediaminetetraacetic acid,propylenediaminetetraacetic acid or mixtures thereof which comprisescombining one or more of said chelates with a biologically pure cultureof Agrobacterium sp.

In another method aspect, the present invention is directed to a methodof recovering iron from an aqueous solution containing ferric chelatesof aminopolycarboxylic acid which method comprises

a) adding a biologically pure culture of Agrobacterium sp. to an aqueoussolution containing one or more ferric chelates of aminopolycarboxylicacid;

b) maintaining the aqueous solution at a pH of about 8 or less at atemperature and for a period of time sufficient for iron to precipitatefrom solution; and

c) recovering said iron.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(a) and 1(b) illustrate characteristics of the growth ofAgrobacterium sp. (ATCC No. 55002) in shakeflasks with sodiumferric-EDTA. Solutions containing basal medium (described below), 35 mMsodium ferric-EDTA and 100 mM phosphate were adjusted to pH 6.2 and 7.4by the addition of HCl and NaOH respectively. The incubations proceededin shaker-incubators at 29° C. In FIG. 1(a), the optical densities at600 nm are plotted for aliquots diluted in 0.1N HCl with the plottedvalues corrected for the dilution factor. In FIG. 1(b), ferric-EDTAconcentration is plotted in millimolar units and the measured pH isgiven above the data points.

FIGS. 2(a) and 2(b) illustrate results obtained from incubation ofAgrobacterium sp. (ATCC No. 55002) at the 1 liter scale with pHcontrolled at 7.4 and temperature maintained between 29°-31° C. Theinitial broth was prepared to contain 35 mM sodium ferric-EDTA and 100mM phosphate in basal medium. In particular, FIG. 2(a) illustratesChemical Oxygen Demand (COD) measurements at different times during theincubation. Specifically, aliquots of the whole broth were taken for CODmeasurements (COD with cells) and after centrifugation at 13000 g toremove cell mass and any chemical precipitate (COD supernatant). FIG.2(b) illustrates the concentration of soluble iron (i.e., in solution),of ammonia and of ferric-EDTA at different points of the incubation.

FIGS. 3(a) and 3(b) illustrate results (including carbon and nitrogenmass balance) from a large scale (7.2 liter) incubation of Agrobacteriumsp. (ATCC 55002) with ferric-EDTA. The COD values were obtained foraliquots after centrifugation to remove cells. The conversion of carbonand nitrogen from ferric-EDTA to CO₂ and ammonia are plotted aspercentages of the maximum values possible from the initialconcentration of ferric-EDTA measured at the time of inoculation. Thepercentage of COD removed is calculated by subtracting that remaining insolution from that in the initial broth. Neither COD nor ammonia valueswere corrected for evaporation.

FIG. 4 illustrates the effect of initial pH and ferric-EDTAconcentration on the growth of Agrobacterium sp. (ATCC 55002). Testtubes contained basal medium, phosphate buffer (100 mM) and sodiumferric-EDTA at the concentrations indicated in the Figure. Test tubeswere inoculated at a calculated initial Optical Density (OD) of 1.0 froma concentrate of freshly harvested and washed cells and were incubatedat 30° C.±1° C. for 48 hours. Error bars indicate the standard deviationfor triplicate experiments (pH 7.4) and duplicate experiments (pH 6.2).Controls were not run in replicate.

FIG. 5 is an electron micrograph of Agrobacterium sp.,platinum/palladium shadowed, grown on tryptic soy broth agar plates.

FIG. 6 is an electron micrograph of Agrobacterium sp.,platinum/palladium shadowed, grown on ferric-EDTA agar plates. The smalldark patches indicate localized high-density regions on the surface ofthe bacterium.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A biologically pure culture of Agrobacterium sp., ATCC No. 55002, wasisolated from a treatment facility receiving industrial waste containingthe ferric chelate of ethylenediaminetetraacetic acid (ferric-EDTA) andis capable of degrading ferric chelates of aminopolycarboxylic acids atrates and substrate concentrations significantly greater than anypreviously reported. This microorganism is an aerobic, gram negative rodof approximately 1 micron in diameter and approximately 2-3 microns inlength. For purposes of this application, a biologically pure culture ofAgrobacterium sp. is a culture which contains cells of one kind, allprogeny of a single ancestor or identical ancestors as well as mutantsand variants thereof having the distinguishing features of Agrobacteriumsp.

It has been discovered that the isolated Agrobacterium sp. microorganismmetabolizes ferric-EDTA as the sole carbon source with a rate ofconsumption as rapid as 24 mM/day. Preferably, in aqueous solutions, theferric-EDTA concentrations can range from about 1 mM or less up to about150 mM or more and more preferably from about 3 mM up to about 80 mM.Thus, the microorganism of the invention is capable of degradingferric-EDTA at a rapid rate and at high substrate concentrations. Asmetabolism proceeds, carbon dioxide and ammonia are produced, pH rises,and iron is precipitated. The precipitated iron can be recovered by anyconventional means such as filtration, centrifugation, etc. The isolatedmicroorganism, Agrobacterium sp., also metabolizes the ferric chelatesof other aminopolycarboxylic acids, such as propylene diaminetetraaceticacid (PDTA) and the like.

For purposes of this application, the term "ferric chelates ofaminopolycarboxylic acid" means those aminopolycarboxylic acids havingtwo or more amino group and 3 or more carboxylic acid groups and whichare capable of forming chelates with Fe⁺³. Aminopoly-carboxylic acidswhich may be degraded (as the ferric chelate) by the microorganism ofthe invention include, for example, ethylenediaminetetraacetic acid(EDTA), propylenediaminetetraacetic acid (PDTA), and the like.

A biologically pure culture of the newly discovered microorganism,Agrobacterium sp., can be used in a process for the removal of ferricchelates of aminopolycarboxylic acids. Preferably, the process isconducted in an aqueous solution wherein the aqueous solution ismaintained at a pH of about 8.0 or less. Alternatively, it iscontemplated that the process can be conducted in a landfill, or othersimilar site, which has been contaminated with ferric chelates ofaminopolycarboxylic acids. As before, it is preferable for the pH of themedium to adjusted to a pH of about 8.0 or less.

When conducted in an aqueous solution, it is contemplated that theprocess is useful with a wide variety of aqueous solutions such asindustrial waste solutions, cleaning solutions, among others.Additionally, the aqueous solution employed herein should not containbiocidal effective amounts of a biocide. In a preferred embodiment, inorder to enhance the degradation rate of ferric chelates, the aqueoussolution should contain less than inhibitory effective amounts ofinhibitors of Agrobacterium sp., i.e., an amount of an inhibitory agentwhich causes a 50% decrease in ferric-aminopolycarboxylic acidmetabolism.

In preferred embodiments of the process of the invention a mineral andbiotin stock is added to the aqueous solution. Specifically, the saltsammonium chloride, magnesium sulfate and calcium sulfate can be used atconcentrations of 5 mM, 0.1 mM and 0.07 mM respectively. In theseembodiments, certain trace mineral salts are added to the aqueoussolution from which the ferric chelates are to be degraded in order toprovide an improved media for growth of the microorganisms. These saltsmay include H₃ BO₃, MnSO₄.7H₂ O, ZnSO₄.7H₂ O, CuSO₄.H₂ O, (NH₄)₆ Mo₇O₂₄.H₂ O, CoSO₄.7H₂ O. The trace salts preferably are present in amountsof less than about 25 micromolar. Specifically, these salts can beemployed at concentrations of 0.025 mM, 0.003 mM, 0.002 mM, 0.00035 mM,0.0002 mM and 0.00004 mM respectively. Biotin may also be added to thesolution and typically is present in amounts of about 1 to about 10micromolar. Specifically, biotin can be employed at a concentration of 2micromolar. Aqueous solutions containing the specific concentrationsrecited above of the salts, trace mineral salts, and biotin aresometimes referred to herein as "basal medium".

Other ingredients which may be present in the aqueous solution includecompatible buffers, i.e., buffers which are not toxic to themicroorganism, such as sodium phosphate, potassium phosphate, and thelike. A sufficient amount of buffer is added so as to maintain the pH ofthe aqueous solution in the desired range. The preferred buffer for theprocess of the invention is phosphate buffer, e.g., a 50/50 molar mix ofsodium and potassium salts at a total phosphate concentration of from 25to 200 mM. However, any compatible buffer or buffers can be used if acidis periodically added to maintain the pH at a level of below about 8.0.Preferably, the pH of the aqueous solution is maintained between about 6and 8 and preferably, the buffer or buffers are added to the aqueoussolution generally are present in amounts of about 25 mM to about 200mM.

In order to maintain the aqueous solution at a pH of about 8.0 or less,acid may be added to the solution. The acid can be any acid which doesnot affect the degradation of the ferric chelates by the Agrobacteriumsp. microorganism. Such acids include, HCl, H₂ SO₄, and the like.Preferably, HCl is used. The acid is added periodically to keep the pHbelow about 8.0.

The temperature of the aqueous solution containing both the metalchelate and Agrobacterium sp. is not critical provided that atemperature is employed which is compatible with the microorganism.Accordingly, any temperature may be employed which is sufficient toallow the microorganism to degrade ferric chelates. In preferredembodiment, the temperatures employed range from about 21° C. to about37° C. and even more preferably, from about 29° C. to about 31° C.

The following examples illustrate the isolation, identification andtesting of Agrobacterium sp. and are provided to further illustrate theinvention, but are not meant to limit the scope of the claims in anyway.

EXAMPLES

The procedures described below were used to evaluate the microorganismof the invention.

Bacterial isolations.

Samples of sludge were obtained from an aerated secondary wastetreatment facility that had been receiving wastes containing ferric-EDTAfor several years. Samples were collected in plastic bottles,transported on ice and then held at 4° C. prior to use. An enrichmentmedium (EDTA EM, See Table 1) was inoculated with 100 microliter ofsample and incubated at 30° C. for two weeks. Growth from this initialenrichment was streaked onto agar plate medium (agar solidified EDTA EM)and incubated at 30° C. until growth was observed. Bacterial coloniesfrom these plates were repeatedly transferred and tested for growth on astringent agar medium (EDTA M1 and M2, see Table 1) to select forcolonies using ferric-EDTA as sole carbon source. Isolated colonies onthese media were then transferred to aerobic liquid culture mediacontaining ferric-EDTA as sole carbon source (EDTA G1, see Table 1).

                  TABLE 1                                                         ______________________________________                                        Enrichment, Isolation and Growth Media.sup.a                                         Concentration (mM) in Media                                            Ingredient                                                                             EDTA EM   EDTA M1   EDTA M2 EDTA G1                                  ______________________________________                                        Citrate- 50 (pH 5.5)                                                          phosphate                                                                     buffer                                                                        MES.sup.b          50 (pH 6)                                                  Phosphate                    50 (pH 6)                                                                             100                                      buffer                               (pH 7.4)                                 Na.sub.2 S.sub.2 O.sub.3.                                                              40        40        40                                               5H.sub.2 O                                                                    Ferric-   5.sup.c   5.sup.c   5.sup.c                                                                               35.sup.d                                EDTA                                                                          NaHCO.sub.3                                                                            24                                                                   Agar     17 g/L.sup.e                                                                            17 g/L    17 g/L                                           ______________________________________                                         .sup.a All media employed the mineral and biotin base described at page 1     hereinabove as the basal medium.                                              .sup.b [NMorpholino]ethanesulfonic Acid                                       .sup.c Ammonium salt.                                                         .sup.d Sodium salt.                                                           .sup.e No agar in liquid enrichment medium EDTA EM                       

In all studies, media containing ferric-EDTA were sterilized prior toincubation with Agrobacterium sp. In some cases, this was done byfiltration through 0.2 micron filters after addition of all componentsand after adjustment of pH. In other cases, the total broth was notfiltered, rather stock solutions were filter sterilized individually andthen added together at which time the pH was adjusted using anethanol-washed pH electrode to maintain sterility. In still other cases,the stock solutions, with the exception of biotin, were heat sterilizedin situ. Then a stock solution of FeCl₃ (0.1M) was prepared forsupplementation of iron at 1 mM. The biotin and FeCl₃ stocks were filtersterilized and added separately. Media which was inadvertentlycontaminated with organism(s) other than Agrobacterium sp. did notsupport extensive degradation of ferric-EDTA and, accordingly, mediacontaining such other organism(s) are not preferred.

Cell Harvest and Storage.

After growth in ferric-EDTA media, cells were harvested bycentrifugation at 4° C. Broth was first centrifuged at 4200 g for 20minutes and then the supernatant fluid was decanted and centrifuged at13000 g for 20 minutes. The cell pellet was resuspended in steriledistilled water and centrifuged again at 13000 g to wash the cells. Theprecipitated cell pellet was resuspended in sterile distilled water atan optical density >5 at 600 nm. For long term storage, glycerol wasadded (10% by volume) to the suspension and aliquots (2 ml) stored in aliquid nitrogen freezer.

The procedure given above for washing the inoculum before storage orinoculation of fresh media was adopted to produce conditions which wouldlead to rapid degradation. In several instances when unwashed inoculawere used, growth in fresh media either lagged or was considerablyslower than that normally observed. Washing removes much of theprecipitate as determined by light microscopy.

Alternatively, the cells can be grown in a complex trypticase/soy broth(from BBL) maintained at 29°-31° C. for 24 hours. The cells so grown arestill effective in degrading ferric-EDTA when added to an aqueoussolution containing ferric-EDTA.

For inoculation, cells from frozen stocks were added or cells wereharvested from seed flasks in the later stages of growth. When freshlyharvested, cells were prepared as above except that after the initialcentrifugation at 13000 g, the cells were suspended in water or freshmedia as the inoculation medium.

Electron Microscopy.

Cultures were fixed with 2% glutaraldehyde in 0.1M cacodylate buffer for2 minutes and then dropped onto Formvar coated grids, dried and vacuumcoated with platinum/palladium at an angle of 14 degrees.Photomicrographs were obtained with rotary shadowing on a JEOL-100 CXelectron microscope.

Strain Identification.

Representative samples were subcultured on blood agar and McConkey agarplates. After incubation, samples were prepared for identification byVitek AMS using the GNI Card (Vitek Systems, Inc., Hazelwood, Mo. 63042)and API 20E using the API Rapid NFT (API Analytab Products, Plainview,N.Y. 11803) systems for the identification of bacteria by the analysisof biochemical reactions. In addition, gas chromatographic analysis ofwhole cell fatty acids as described in Drucker, Methods in Microbiology,Vol. 9, J. R. Norris, Ed. Academic Press, London, p. 51-125, was alsoused to confirm the identification.

Ferric-EDTA Assay.

Cells were separated from samples prior to the determination offerric-EDTA by centrifugation in an Eppendorf Model 5415 centrifuge at14,000 rpm for 2 minutes. Samples were then diluted with water orinitial mobile phase such that the maximum ferric-EDTA concentration ofthe diluted samples was less than 0.9 mM.

HPLC analysis were performed using a modular Waters Associates system(Millipore Corp., Milford, Mass. 01757) equipped with a WISP 712 sampleprocessor for injection of samples, two Waters 510 pumps for mobilephase delivery, a Waters 490 multiwavelength detector set at 360 nm fordetection of the ferric-chelate and a Waters 840 data system fordisplay, storage, plotting and analysis of the chromatograms.

The chromatographic separations were obtained after injection of 30microliters of sample onto a commercially available, 15 cm long by 4.6mm i.d. reversed-phase HPLC column (LC18 from Supelco, Inc., Bellefonte,Pa. 16823). Isocratic elution at 1.0 milliliter/minute with an initialmobile phase consisting of 1 g/liter ammonium acetate, 0.5 g/liter PDTA,1.5 milliliter/liter glacial acetic acid, 0.5 milliliter/liter ammoniumhydroxide, 0.1 milliliter/liter triethylamine and 5 milliliter/literacetonitrile in deionized water caused elution of ferric-EDTA at 2minute retention time. After 2.5 minutes from the injection, astep-gradient to a final mobile phase consisting of 0.5 g/liter PDTA,0.1 milliliter/liter triethylamine and 600 milliliter/liter acetonitrilein deionized water for 5.5 minutes washed highly retained componentsfrom the column. A further step gradient to the initial mobile phase for4 minutes was used to reequilibrate the column.

The instrument was calibrated daily by the injection of a series ofstandards of ammonium ferric-EDTA in unbuffered, deionized waterencompassing the concentrations from 0.03 to 0.9 mM. Linear-leastsquares regression analysis of the area responses of the standards wasused to obtain slope and intercept values (r-squared typically >0.996)which were then used to obtain quantitative values for samples from themeasured area. Precision and accuracy were estimated to be better than5% relative over the calibration range.

Ammonia/Ammonium Assay.

Total ammonia was determined using an Orion Model 95-12 ammoniaelectrode (following the procedures set forth in the Orion Model 95-12instruction manual, Orion Research Inc.).

COD Assay.

For the estimation of COD in samples, the test tube colorimetricprocedure of the Hach Chemical Co. (Hach Co., Loveland, Colo. 80539),which is similar to Method 508C in "Standard Methods for the Examinationof Water and Wastewater" American Public Health Association, Washington,D.C., 16th Edition, pp. 532-538 (1985) was used.

Iron Assay.

For the estimation of total iron in aqueous samples, a modification ofStookey's ferrozine procedure described in Stookey, Anal. Chem., 42:779-781 (1970) was used. To release the iron cation from chelatorspresent, including ferric-EDTA, samples were first digested using aprocedure adapted from Method 302E in "Standard Methods for theExamination of Water and Wastewater", American Public HealthAssociation, Washington, D.C., 16th ed. p. 148 (1985). A 50-microlitersample was added to an acid-cleaned, thick-walled, screw-capped testtube (equivalent to Hach COD test tubes), followed by 50 microliters ofconcentrated nitric acid and 100 microliters of concentrated sulfuricacid. The test tube was tightly-capped and heated to reflux at 150° C.for 15 minutes in a Hach Reactor Plate. After cooling, the tubes wereuncapped and replaced in the reactor for a maximum of 10 minutes, butnot long enough for the test tube to become thoroughly dry. Aftercooling, 200 microliters of acid-ferrozine reagent (0.514 g ferrozine,10 g hydroxylamine hydrochloride, and 50 milliliters of concentratedhydrochloric acid dissolved and diluted to 100 ml with distilled water)was added. The contents were mixed using a vortex mixer and then 1.5milliliters of buffer (40 g ammonium acetate and 35 milliliters ofconcentrated ammonium hydroxide diluted to 100 milliliters withdistilled water) was added to adjust the pH between 5 and 9. The samplewas then diluted with 5.4 milliliters of distilled water.

Standards encompassing the concentration range from 20 to 1000 mg/Ltotal iron were prepared from pure ferric ammonium sulfatedodecylhydrate. Standards were treated as the samples were. From theabsorbance of the standards measured at 562 nm in a spectrophotometer(Hach DR/3) and concentrations of the standards, a linear least squarescalibration was constructed. At concentrations of total iron greaterthan 50 mg/L, the observed precision and accuracy using samplescontaining known concentrations of ferric-EDTA were better than 5%.

Example 1--Bacterial Identification

The identification of the isolated strain as Agrobacterium sp., agram-negative aerobe, was made on a Vitek AMS (Vitek Systems, Hazelwood,Mo. 63042-2395) using a Vitek GNI Card (gram negative identificationcard; product no. 51-1306) with a probability of 99%. The biochemicaltests employed for the identification and the results for the isolatedstrain are presented in Table 2. The identification was also confirmedby an API Rapid NFT strip (API Analytab Products, Plainview, N.Y. 11803;product no. 8886-500951) with a probability of 99.9%. The biochemicaltests employed for the identification and the results for the isolatedstrain are presented in Table 3. Gas-chromatographic analysis of thefatty acid composition of the strain also confirmed the genera asAgrobacterium.

                  TABLE 2                                                         ______________________________________                                        Vitek GNI Test Results for Isolated Strain                                    Biochemical Test:                                                                         Reaction Biochemical Test:                                                                            Reaction                                  ______________________________________                                        Dp-300      -        Sorbitol       -                                         Glucose     +        Sucrose        -                                         Acetamide   -        Inositol       -                                         Esculin     +        Adonitol       -                                         Plant Indican                                                                             +        p-Coumaric     -                                         Urea        +        H.sub.2 O      -                                         Citrate     -        ONPG           -                                         Malonate    -        Rhamnose       -                                         Tryptophan  -        Arabinose      +                                         Polymyxin B -        Glucose (ferment.)                                                                           -                                         Lactose     +        Arginine       -                                         Maltose     +        Lysine         -                                         Mannitol    +        Ornithine      -                                         Xylose      +        Oxidase        +                                         Raffinose   -                                                                 ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        API Rapid NFT Test Results for Isolated-Strain                                Carbon Source:                                                                            Growth   Biochemical Test:                                                                            Reaction                                  ______________________________________                                        D-Glucose   +        Nitrate Reduction                                                                            +                                         L-Arabinose +        Tryptophanase  -                                         D-Mannose   +        Glucose fermentation                                                                         -                                         D-Mannitol  +        Arginine dihydrolase                                                                         -                                         N-acetyl-D-gluco-                                                                         +        Urea           -                                         samine                                                                        Maltose     +        Esculin        +                                         D-Gluconate +        Gelatinase     -                                         Caprate     -        Beta-galactosidase                                                                           +                                         Adipate     -                                                                 Malate      +                                                                 Citrate     -                                                                 Phenylacetate                                                                             -                                                                 Oxidase     +                                                                 ______________________________________                                    

In view of the above results, the microorganism was tentativelydetermined to be Agrobacterium radiobacter. In addition, the isolate wastyped independently (American Type Culture Collection, Rockville, Md.)as an Agrobacterium sp. with distinction between biovar 1 and biovar 2not determined. Accordingly, this microorganism is referred to herein asAgrobacterium sp.

Example 2--Growth studies

Growth studies were carried out to determine various characteristics ofthe microorganism. For these studies, the basal medium recited at page 9hereinabove was employed as well as 100 mM phosphate buffer, and 35 mMsodium ferric-EDTA in Fernbach flasks. Incubation was at 29° C. in ashaker-incubator. Samples were taken periodically and the pH,ferric-EDTA concentration and optical density (OD) at 600 nm weredetermined. The pH was measured using an Orion combination electrode anda Corning pH meter. OD was used as a measure of bacterial growth.Measurements were made by dilution in 0.1N HCl to dissolve the ironprecipitate which formed during degradation and which interfered inreading OD due to bacterial growth.

The increase in OD observed during growth in the Fernbach flasks isshown in FIG. 1(a).

In FIG. 1(b), the concentration of ferric-EDTA in the flasks is plottedalong with the measured pH. The plots demonstrate that as the opticaldensity increases in all three flasks, the concentration of ferric-EDTAdecreases and the pH increases. When pH does not increase, no metabolismof ferric-EDTA is observed in any of the flasks. Conversely, if the pHis not constrained from increasing above pH 8, the disappearance offerric-EDTA stops. The apparent increase of ferric-EDTA concentration inthe flask without pH adjustment is caused by evaporation afterferric-EDTA metabolism has ceased (pH >8.1). No correction forevaporation from any of these flasks was made. The data contained inthese figures demonstrates that Agrobacterium sp. grows whileferric-EDTA is being degraded and when degradation ceases, so doesgrowth.

Example 3--Incubation at the 1 liter scale

The isolate was grown at the 1 liter scale in a 2 liter incubator toenable the control of pH. The initial incubator broth was 35 mM insodium ferric-EDTA, 100 mM in phosphate, and contained the basal mediumdescribed above. In addition, FeCl₃ was added to 1 mM finalconcentration. For inoculation of the incubator, the strain was grown ina shakeflask at 31° C. in the same media as above for 24 hours. Thecells from the shakeflask were separated from the broth bycentrifugation for 20 minutes at 13000 g. These were resuspended in 200mL of fresh broth and added to the incubator to bring the initial totalvolume to 1 liter.

Air was passed into the Multigen fermentor (New Brunswick ScientificInc., Edison, N.J. 08818) through a 0.2 micron sterile filter at aflowrate of 1 liter/minutes. The fermentor (incubator) was stirred witha magnetically driven propeller shaft at 500 rpm. The hydrogen ionconcentration was monitored and controlled at between pH 7.4 and 7.7 bythe automatic addition of HCl. Temperature of the incubator wasmaintained at 30° C.±1° C.

The results are plotted in FIGS. 2(a) and 2(b). The decrease inferric-EDTA concentration observed as the incubation proceeded wasaccompanied by a parallel decrease an iron concentration in solution. Asthe substrate was being removed from aqueous solution, the ammoniumcontent increased. In addition, COD fell, but not in proportion to thedecrease in ferric-EDTA concentration (93%). For samples measured aftercentrifugation to remove the cell mass and precipitate, COD fell from8880 to 2640 mg 0₂ /liter (70% decrease); for samples measured withcells and precipitate still present, COD fell from 8870 to 3392 mg 0₂/liter (62% decrease).

In addition, selected samples were diluted and plated on TSA forbacterial counting. The live bacterial count began decreasing between 46and 70 hours after the beginning of the experiment. This coincidesclosely with breaks in the slopes of the curves.

Example 4--Inhibition

The inhibitory effect of several factors on ferric-EDTA metabolism wasinvestigated. These experiments were carried out in test tubes using astandard set of conditions: 100 mM phosphate buffer (pH 7.4); basalmedium described above; 35 mM in ferric-EDTA; inoculation to an opticaldensity of 1 at 600 nm; initial volume of 3.5 mL; incubation at 29°-31°C. for 1 to 4 days in a test tube shaker incubator. Concentrates of thefactors to be tested for inhibition were added at 4 to 6 differentlevels to different sterile test tubes with the rest of the volumeprovided by the addition of deionized water.

The data on the inhibitory effects of several components are summarizedin Table 4. The concentrations in the table are those which caused a 50%decrease in metabolism of ferric-EDTA over the time of the experimentscompared to controls containing none of the factor tested.

                  TABLE 4                                                         ______________________________________                                        Inhibition of Strain ATCC #55002                                              Conc. (mM)                                                                    Causing 50% inhibition                                                                       Ferric-EDTA Metabolism                                         Factor         Inhibition                                                     ______________________________________                                        Ferric-EDTA     80                                                            Hepes          <5                                                             Mops           <5                                                             Triethanolamine                                                                               15                                                            Tris            5                                                             Ionic Strength (NaCl)                                                                        540                                                            Ammonium       130                                                            ______________________________________                                         Hepes -- 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid                    Mops -- 3(4-Morpholino)propanesulfonic acid                                   Tris -- tris(hydroxymethyl)aminomethane                                  

In addition to inhibition by the specific chemicals listed above,unidentified metabolite(s) of ferric-EDTA degradation by Agrobacteriumsp. are pro-duced during degradation and lead to product inhi-bition.Also, this microorganism is inhibited by one or more components found inphotoprocessing solutions.

Example 5--Chelators as growth substrates

A number of metal chelators were tested as substrates for growth. Theseexperiments were carried out in test tubes which were placed in abenchtop shaker for 24 days at 30° C. The medium was the same as thatdescribed above for growth inhibitors except that the initial phosphatebuffer concentration was 71 mM and the inoculum was calculated toproduce an initial optical density of 0.4. Concentrates of the organicchelators were added to the replicate test tubes at final concentrationsof 4 and 20 mM. For samples containing metals, a molar amount equivalentto that of the chelator was added from concentrates of FeCl₃, NiCl₂ orCuSO₄. After addition of all components except the inoculum, the sampleswere adjusted to pH 7.4 with either dilute HCl or KOH. A duplicate setof test tubes was prepared as above, but with the addition to allsamples of 0.25 mL of a 2.5 g/L solution of HgCl₂ to prevent bacterialgrowth.

Growth in the samples after 24 days was assessed by comparing theoptical densities of sample tubes with those in the controls withmercuric chloride added (Table 5). Those substrates which had an opticaldensity five-fold greater than the respective controls at bothconcentrations tested were considered positive for growth.

                  TABLE 5                                                         ______________________________________                                        Growth Substrates for Agrobacterium sp.                                       Substrate Growth   Substrate        Growth                                    ______________________________________                                        EDTA      -        Fe.sup.3+ EDDA   -                                         Fe.sup.3+ EDTA                                                                          +        Ethylene diamine (ED)                                                                          -                                         Ni.sup.2+ EDTA                                                                          -        Fe.sup.3+ ED     -                                         Cu.sup.2+ EDTA                                                                          -        TEA (triethanolamine)                                                                          -                                         NTA       -        Fe.sup.3+ TEA    -                                         Fe.sup.3+ NTA                                                                           -        Citrate          -                                         Ni.sup.2+ NTA                                                                           -        Fe.sup.3+ Citrate                                                                              -                                         Cu.sup.2+ NTA                                                                           -        Propionate       -                                         PDTA      -        Fe.sup.3+ Propionate                                                                           -                                         Fe.sup.3+ PDTA                                                                          +        Acetate          -                                         Ni.sup.2+ PDTA                                                                          -        Fe.sup.3+ Acetate                                                                              -                                         Cu.sup.2+ PDTA                                                                          -        Lysine           -                                         IMDA      -        Fe.sup.+3 Lysine -                                         Fe.sup.3+ IMDA                                                                          -        EDDA             -                                         ______________________________________                                         IMDA = iminodiacetic acid                                                     NTA = nitrilotriacetic acid                                                   EDDA = ethylenediamine diacetic acid                                     

Example 6--Incubations at the 7.2 liter scale

In order to control aeration, mixing and pH better and to determine thecarbon and nitrogen mass balance, incubations were run at volumes of 7.2liters in computer controlled 14 liter Chemap CF 3000 incubators (ChemapAG, Volketswil, Switzerland) maintained at 28° C.

Two 6 liter batches of ferric-EDTA medium containing basal medium(described above) were inoculated with freshly harvested cellsconcentrated in 1.2 liters of the same basal medium. One of theincubations also received a steady feed of 250 g/L sucrose and 250 g/Lyeast extract at a constant rate of 0.2 g/min. This feed composition waschosen based on previous work with the cultivation of Agrobacteria inHofer, J. Bacteriology, 41: 193-224 and Lippincott et al, TheProkaryotes, Vol. 1, Starr et al, Eds. Springer-Verlag, pp. 842-845.

FIG. 3(a) is a graphical representation of the cumulative conversion offerric-EDTA to CO₂ and to NH₃ as percentages of the maximum possiblefrom ferric-EDTA EDTA in the incubation with only ferric-EDTA as thecarbon source. In addition, the percentage decrease in COD is alsoplotted. All three curves parallel each other and further demonstratethat ferric-EDTA is efficiently minerialized. FIG. 3(b) plots therelative concentrations of soluble iron and ferric-EDTA during thecourse of the incubation supplied solely with ferric-EDTA as carbonsource.

In the incubation with additional carbon and/or nitrogen sources(sucrose/yeast), degradation of ferric-EDTA and growth of themicroorganism was less vigorous, although substantial degradation didoccur. In particular, for the same duration of incubation, 37% of theferric-EDTA was degraded as opposed to more than 85% for the case shownin FIG. 3(b). Accordingly, substantial degradation of ferric-EDTA inwaste streams containing non-biocidal carbon sources can be expected.

Example 7--Effect of ferric-EDTA concentrations on Degradation byAgrobacterium sp. (ATTC 55002)

To determine the maximum initial ferric-EDTA concentration that theisolate would tolerate, a series of test tube incubations using thebasal medium (described above) buffered with phosphate at 100 mM werecarried out for 48 hours at 30° C.±1° C. The initial ferric-EDTAconcentration was varied from 2.9 to 140 mM. The values for theconcentration removed (calculated by subtracting the concentrationremaining at 48 hours from the concentration initially added) areplotted in FIG. 4. No pH adjustments were made during this experiment.

The extent of degradation was related to the initial pH. At the lowerinitial pH, for all concentrations tested more ferric-EDTA was degraded.The degradation proceeded to consume 48 mM of substrate at an initial pHof 6.2 and 28 mM at pH 7.4. In both sets of data, the maximumdegradation extended to the highest concentrations tested.

At the higher initial hydrogen ion concentration (pH 6.2), ferric-EDTAis metabolized at a limiting rate of 24 mM/day while at the lowerinitial hydrogen ion concentration (pH 7.4), the maximum rate is 15mM/day.

Example 8--Morphology

Cells of the isolate were grown on agar plates containing either trypticsoy broth or ferric-EDTA as sole carbon source (see Table 1, medium Glwith 17 g/L agar). Electron micrographs of several aliquots of eachculture were taken as described above. Examples of these are reproducedin FIG. 5 (tryptic soy broth) and FIG. 6 ferric-EDTA. In both cases theculture consisted of rods from approximately 2-3 microns long byapproximately 1 microns in diameter. Only on microbes grown onferric-EDTA were small dark patches noted.

As described in the Examples, a culture of Agrobacterium sp. can be usedin a process for the degradation of ferric chelates ofaminopolycarboxylic acids. The process preferably employed on aqueoussolutions containing such ferric chelates and is particularly suitablefor degrading ferric-EDTA, ferric-PDTA, or mixtures thereof from aqueoussolutions. The rate and extent of degradation are dependent on the pH,or hydrogen ion concentration. As biodegradation proceeds and suchferric chelates are consumed, both cell mass and pH increase. If thehydrogen ion concentration is allowed to fall much below 10⁻⁸ (pHgreater than about 8), degradation and growth cease. By the addition ofacid periodically to constrain pH from rising above about 8, moreextensive degradation occurs.

Without being limited to any theory, in these experiments it is believedthat the source of at least some of the increase in pH can be accountedfor by the production of ammonia which is observed to increase inconcentration as the ferric-EDTA is metabolized. Although ammonia itselfinhibits growth of the microorganism (Table 4), significant inhibition,i.e., 50% or more, appears at a higher concentration than theconcentration produced in these incubations.

The degradation of the aminopolycarboxylic acids by the Agrobacteriumsp. is primarily biological, as shown by the Examples. In uninoculatedcontrols, as in those summarized in FIG. 4, neither optical density norpH significantly increased as the incubation proceeded, nor was asignificant decrease of ferric-EDTA concentration observed. Conversely,in inoculated samples, as degradation proceeded, the decrease inferric-EDTA concentration during incubation was always accompanied by anincrease in optical density and pH. Finally, the rapid increase inammonia/ammonium concentration and the substantial release of CO₂observed in the inoculated samples demonstrates a relatively completemetabolism of the substrate. It is believed that any influence of lighton the degradation was minimal or nonexistent.

The efficiency of the mineralization of organic carbon from ferric-EDTAdegraded by the microorganism of the invention can be calculated fromthe data plotted in FIG. 3(a). At 3 days, 64% of the initial carbon canbe accounted for by the evolved CO₂. The COD remaining in solution atthis time, after centrifugation to remove cell mass and associatedprecipitate, corresponds to 39% (an overestimate since the measuredvalues for COD were not corrected for evaporation) of that initiallypresent. Even accounting for the maximum potential errors associatedwith both measurements (<10% relative) and the effects of evaporation,less than 20% of the carbon initially present could have been convertedto cells.

The data for the 1 liter incubations confirms the relatively smallamount of cell mass produced from the biodegradation. At the end of theexperiment when the ferric-EDTA concentration remaining in solution was7% of that initially present, the difference between the COD remainingin the total broth (including cells, precipitate, and solublemetabolites) and that remaining in the solution after the cells andprecipitate were removed by filtration (soluble metabolites) was lessthan 10% of that in the initial fresh broth (752 vs 8870 mg O₂ /liter).The difference reflects the maximum oxidizable carbon which could havebeen incorporated into cell mass. Thus, the metabolism of ferric-EDTA bythis microorganism leads primarily to mineralization and not toincreased cell mass.

In addition to the CO₂ and cell mass, an unidentified degradationproduct or products accumulates in solution. This product(s) is evidentfrom the residual COD present in the solution at the end of theincubation after removal of cells and precipitates. The Agrobacteriumsp. (ATCC 55002) leads to degradation of 90% or more of the initialferric-EDTA present. However, the COD reduction only approaches 70%.Thus 20-30% of the carbon initially present as ferric-EDTA remains insolution as unidentified metabolites at the end of the incubation. Thesemetabolites can be inhibitory to the growth of the microorganism.

Although not wishing to be bound by any theory, it is believed that themicroorganism of the invention has a requirement for iron. This wasindicated by some preliminary growth studies. In some of these avariable and often long lag time (3 days) was observed when attemptingto cultivate the strain in ferric-EDTA media which contained a largeexcess of EDTA. In the normal procedure for preparing media as describedabove, ferric-EDTA was dissolved in a solution consisting of basalmedium and phosphate buffer. After all components were dissolved, the pHof the media was adjusted. As the pH was increased, a red-orangeflocculent precipitate characteristic of iron hydroxides formed. Themedia were then filter sterilized which removes the precipitated ironand leaves an excess of EDTA in solution. The excess becomes greater ifthe pH is increased further. In fact, in order to ensure minimal lagphase in the larger scale incubations 1 millimolar ferric chloride wasadded to the sterile-filtered incubator media to replace some of theiron removed by the filtration. This procedure consistently producedminimal lag times of less than a few hours.

Besides a deficit of iron, lag times were also found to be related totreatment of the inoculum. When an inoculum was harvested from spentferric-EDTA media by centrifugation without washing of the cells, it wasobserved that, in addition to the cells, a considerable amount ofchemical precipitate was present. It is believed that the precipitate isan insoluble iron salt which forms after the strong chelator, EDTA, hasbeen degraded. In some experiments, if most of the precipitate was notremoved from the inoculum by washing as noted above, slow growth wasobserved in subsequent incubations.

The marked preference of this microorganism forferric-aminopolycarboxylic acids and its effectiveness at relativelyhigh substrate concentrations makes it useful for waste remediationstrategies. In uses as a cleaning, decontamination or descaling agent, alarge proportion of the EDTA or other aminopolycarboxylic acid used isbelieved to become chelated with a heavy metal, in many cases iron. Ifsufficient excess iron is present in, or added to, an aqueous system toforce the equilibrium of the non-iron metal chelates to the ferricchelate, the microorganism of the invention can be used in thatenvironment.

Accordingly, another aspect of the present invention relates to a methodfor the removal of metal chelates of aminopolycarboxylic acid from anaqueous solution containing such chelates wherein the metal is any metalcation, other than Fe⁺³, capable of chelating with saidaminopolycarboxylic acid which method comprises:

a) adding a sufficient amount of a ferric salt to the aqueous solutioncontaining said metal chelates so that a substantial portion of saidmetal chelates become ferric chelates; and

b) adding Agrobacterium sp. (ATCC 55002) to said solution whilemaintaining the pH of said solution at about 8 or less.

In general, sufficient amount of a ferric salt is added so that at leastabout 50% of the metal chelate and preferably, at least about 80% andmore preferably at least about 95% is converted to ferric chelates.Suitable ferric salts for use herein include any ferric salt which issoluble in said aqueous solution including without limitation, ferricammonium sulfate, ferric nitrate, ferric chloride, ferric sulfate, andthe like. Further, in this regard, it is noted that certain ferric saltswhich are insoluble in water but soluble in aqueous acidic solutions maybe employed herein by merely employing an acidic aqueous medium in whichsuch ferric salts are soluble and which the microorganism Agrobacteriumsp. will tolerate.

From the foregoing description, various modifications and changes in theprocess will occur to those skilled in the art. All such modificationscoming within the scope of the appended claims are intended to beincluded therein.

What is claimed is:
 1. A biologically pure culture of the microorganismAgrobacterium sp., ATCC number
 55002. 2. The biologically pure cultureof claim 1, said culture being capable of removing ferric chelates ofaminopolycarboxylic acids from an aqueous solution containing one ormore of said chelates.
 3. The biologically pure culture of claim 2,wherein said aminopolycarboxylic acids are ethylenediaminetetraaceticacid, propylenediaminetetraacetic acid or mixtures thereof.